*3.6. Metatranscriptomics*

Metatranscriptomics is another technique that has been employed in gu<sup>t</sup> microbiota studies leveraging the technological advances in RNA sequencing (RNA-seq) [68]. Metatranscriptomics entails retrieving, sequencing and analyzing total messenger RNA (mRNA) or microRNA (from a microbial ecosystem), to ascertain what genes are expressed within that community [69]. In practice, the retrieved or extracted RNA is converted into a complimentary DNA (cDNA) using a reverse transcriptase and oligo (dT) primers or random hexamers, after which libraries are constructed and sequenced. However, semi direct RNA sequencing, bypassing the conversion of RNA to cDNA, has also been developed. Metatranscriptomics is apt in human gu<sup>t</sup> microbiota exploration, as it shows the real-time functional activities of microbiomes and is better positioned in associating gu<sup>t</sup> microorganisms with host performance [70]. Furthermore, it provides a window through which active pathways are identified, and shows how expressed functions have a role in disease severity and progression [71]. This technology also gives insight into the interaction of the gu<sup>t</sup> microbiota and mucosal immune system, which can help physicians track malfunctions in the host's physiology [68].

### *3.7. High-Throughput Culturing*

Several bacterial culture techniques have been developed overtime making it possible to culture a reasonable number of gu<sup>t</sup> bacteria that had not been cultivated in the past. One such culture technique is culturomics. Culturomics, according to Lagier et al. [72], is a culturing technique that employs multiple culture conditions and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and 16S rRNA gene amplification/sequencing for identification, as shown in Figure 3. Traore et al. [73] isolated 1162 bacteria strains by culturomics in a study that compared the gastrointestinal microbiota of Africans to the West. Goodman et al. [74] reported the use of gu<sup>t</sup> microbiota medium (GMM) with high-throughput anaerobic culturing techniques in combination with metagenomics in characterizing extensive personal human gu<sup>t</sup> microbiota culture collection and manipulation in gnotobiotic mice. Similarly, Lau et al. [49] revealed that using culture-enriched molecular profiling consisting of 66 culture conditions in conjunction with 16S rRNA gene sequencing was able to yield a robust data on the diversity of the microbiota that were present in sampled human feces. High-throughput culture methods have obvious advantages of enhancing the culturability of otherwise 'non-culturable' bacterial population, hence, giving room for the in-depth study of identified species. This technique is quite elaborate requiring specialized laboratories and it is space and time consuming. Harnessing the enormous potentials of high-throughput culturing techniques will provide clinicians with the platform for precise treatment of gu<sup>t</sup> associated diseases resulting from perturbation, since implicated microbiota members can be cultured. Additionally, in the formulation and administration of probiotics, this technique can be useful. Furthermore, the morphology, physiology and biochemistry of individual microorganism can be studied and their response/interaction with drugs easily evaluated, thereby enabling the proper treatment of gu<sup>t</sup> diseases.

**Figure 3.** A stepwise outline of culturomics, enabling the culture of previously uncultured bacterial species in the human gut. Source: Lagier et al. [72] (*Nature Microbiology*, Macmillan Publishers Limited) Licensed under CC BY 4.0.

#### **4. Future Perspectives and Conclusions**

Although research in gu<sup>t</sup> microbiota is still evolving, already available data and methods of exploring this complex ecosystem has helped to sharpen our knowledge and understanding of the microbiome and how it affects human health. Several studies and technologies that have focused on interactions between the gu<sup>t</sup> microbiome and diet have revealed how the introduction of new micro-organisms and their change over time have opened up opportunities for their future intervention, as well as diagnostic tools based on the microbiome. These methods have been used to identify the yet-to-be cultured microbes of the gu<sup>t</sup> microbiota, using the activity of various microbial metabolites

for the purpose of pathogen identification. In addition, gu<sup>t</sup> microbiome studies have been used in fecal microbial transplant for the treatment and correction of gu<sup>t</sup> infections/disorders.

These techniques have therefore been proven to provide efficient information on the gu<sup>t</sup> microbiome and how it evolves over time, thereby generating rich data sets that have been useful for treatment of ye<sup>t</sup> to be identified pathogens, while enabling faster and more accurate diagnosis even from non-invasive sampling. Finally, the gu<sup>t</sup> microbiome research has upped the ante on genomic technologies, because genomes of the gu<sup>t</sup> microbiomes are sequenced to give us a better understanding of the gu<sup>t</sup> microbiome.

**Author Contributions:** Conceptualization, S.S.; Methodology, A.A.; Data curation, S.S., A.A., and T.J.; writing original draft preparation, S.S., A.A., and T.J.; writing-review and editing, A.A. and S.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
